Small interfering RNAs (siRNAs) are produced by the cleavage of double-stranded RNA (dsRNA) precursors by Dicer, a member of the RNase III family of dsRNA-specific endonucleases. Typically, siRNAs result when transposons, viruses, or endogenous genes express long dsRNA or when dsRNA is introduced experimentally into plant or animal cells to trigger gene silencing, a process known as RNA interference (RNAi).
siRNAs were first identified as the specificity determinants of the RNA interference (RNAi) pathway, where they act as guides to direct endonucleolytic cleavage of their target RNAs. Prototypical siRNA duplexes are 21 nucleotide, double-stranded RNAs that contain 19 base pairs, with two-nucleotide, 3′ overhanging ends. Active siRNAs contain 5′ phosphates and 3′ hydroxyls.
siRNAs are typically found in the RNA-induced silencing complex (RISC) that mediates both cleavage and translational control. siRNA duplexes can assemble into RISC in the absence of target mRNA, both in vivo and in vitro. Each RISC contains only one of the two strands of the siRNA duplex. Since siRNA duplexes have no foreknowledge of which siRNA strand will guide target cleavage, both strands must assemble with the appropriate proteins to form a RISC.
It has been observed that both siRNA strands are competent to direct RNAi (Tuschl et al., Genes Dev 13, 3191-3197 (1999); Hammond et al., Nature 404, 293-296 (2000); Zamore et al., Cell 101, 25-33 (2000); Elbashir et al., Genes Dev 15, 188-200 (2001); Elbashir et al., EMBO J. 20, 6877-6888 (2001); Nykänen et al., Cell 107, 309-321 (2001). That is, the antisense strand of an siRNA can direct cleavage of a corresponding sense RNA target, whereas the sense siRNA strand directs cleavage of an antisense target. In this way, siRNA duplexes appear to be functionally symmetric.
The ability to control which strand of an siRNA duplex enters into the RISC complex to direct cleavage of a corresponding RNA target would provide a significant advance for both research and therapeutic applications of RNAi technology.